techniques

Fluorescence Resonance Energy Transfer (FRET) - This technique makes use of two fluorophores, one of which is called the donor and the other of which is called the acceptor. If conditions are right, the excited state energy of the donor will be transferred to the acceptor, resulting in reduced emission of the donor and "sensitized" emission of the acceptor. Three conditions determine the efficiency of FRET: the emission spectrum of the donor must sufficiently overlap the excitation spectrum of the acceptor; the transition dipoles of the two fluorophores must be oriented close to parallel; and the two fluorophores must be between 1 and 10 nm of each other. FRET has been used to detect intermolecular interactions between two molecules where one is labeled with a donor and one is labeled with an acceptor (intermolecular FRET). Placing the donor and acceptor at different positions on the same molecule has been used to detect conformation changes in proteins (Intramolecular FRET). Both these methods have been used to design biosensors for enzyme activity and ligand binding. The LCIF has several microscopes capable of CFP/YFP or GFP/RFP FRET imaging. The Zeiss LSM 510 confocal, the Deltavision RT and the Perkin Elmer spinning disk confocal are recommended for intramolecular FRET. The FLIM/TIRF system in ND7.225 is recommended for intermolecular FRET. For further information on FRET imaging, click here.

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Two- or multi-photon microscopy (2P or MPM) - This technique depends on the inverse linear relationship between energy and wavelength of light. For example, a photon with a wavelength of 900 nm will have exactly half the energy of a photon with a wavelength of 450 nm. For excitation of a fluorophore, it is the energy, not the wavelength that counts. If two 900 nm photons arrive simultaneously, the fluorophore will be excited and will fluoresce just as if it had absorbed a single 450 nm photon. Similarly, a fluorophore with an excitation maximum of 300 nm can be excited by absorbtion of three 900 nm photons. Simultaneous absorption of more than one photon is a very rare event, but it can be achieved at high photon densities. In 2P or MPM microscopy, a near infrared (NIR) laser is focused on a biological specimen on the stage of a fluorescence microscope. Pumping the laser output to produce femtosecond pulses of very high numbers of photons results in significant two-photon absorbtion by fluorophores in the focal volume. Several advantages of using NIR light recommend this microscopic method for thick, turbid or living biological specimens. Unlike most other wavelengths of light, NIR interacts very little with biological material. This means that outside the focal volume there is little photobleaching, photoxicity, autofluorescence or scattering to degrade the sample or the fluorescence image. In addition, the excitation light is easily filtered out from the emission light. Because there is essentially no excitation of fluorophores outside the focal volume, 2P is inherently confocal with no need for a pinhole aperture to screen out out-of-focus signal. The Zeiss LSM510 in the LCIF is equipped for 2P microscopy. For more information on 2P or MPM click here.

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Second Harmonic Generation Imaging (SHG) - This technique allows for imaging of endogenous biological structures throughout specimens several hundred micrometers thick without staining or fluorescent labeling. It is analogous to polarization microscopy, in that highly ordered, birefringent structures are detected with high contrast. SHG imaging has been used to visualize collagen, myosin, and tubulin in mitotic spindles in vivo. The SHG signal arises from second-order polarization of ordered arrays of asymmetric molecules, resulting in emission of light at double the frequency (half the wavelength) of the incident light. Thus, NIR laser excitation produces SHG signals in the visible spectrum. SHG can be combined readily with two photon imaging of GFP. For example, upon excitation at 850 nm, GFP fluoresces at the usual emission maximum of 520 nm and any SHG signal can be detected at 425 nm by a relatively simple modification of the microscope. The Zeiss LSM510 in the LCIF is equipped for SHG microscopy. For more information click here.

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Fluorescence Correlation Spectroscopy (FCS) - FCS involves the statistical analysis of flucutations in the number and intensity of fluorophores in a microscopic volume of a dilute sample. These fluctuations occur spontaneously as fluorophores enter or leave the sample volume or upon reversible association of two or more fluorescent molecules or with flucutations in the microenvironment of the fluorophore while it is resident in the sample volume. FCS has been most widely used to obtain the diffusion coefficient of fluorescent molecules, but newer methods permit determination of aggregation states and conformational dynamics. The highly focussed laser excitation of the confocal microscope is well suited for FCS in live cells and a commercial attachment (Confocor) for the Zeiss LSM 510 is available on campus but is not part of the LCIF. For more information click here.

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Total Internal Reflection Fluorescence (TIRF) - A fluorescent sample on a glass coverslip is illuminated obliquely from below by a laser beam or an arc lamp through a high numerical aperture lens so that the angle of incidence of the illumination is greater than the critical angle for total internal reflection. The illuminating light is completely reflected off the coverslip, but an evanescent wave of electromagnetic energy passes into the sample and excites fluorescence. The evanescent wave attenuates with distance into the sample so only fluorophores within about 50 to 100 nm of the coverslip are excited. Instrumentation for this technique is now commercially available from several microscope companies and is being used to study plasma membrane dynamics, including real time movies of exocytosis in neurons and macrophages. The LCIF has two TIRF microscopes.

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